RNA Template Requirements for Target DNA-Primed Reverse Transcription by the R2 Retrotransposable Element

Abstract

R2 is a non-long terminal repeat-retrotransposable element that inserts specifically in the 28S rRNA gene of most insects. The single protein encoded by R2 has been shown to contain both site-specific endonuclease and reverse transcriptase activities. Integration of the element involves cleavage of one strand of the 28S target DNA and the utilization of the exposed 3 hydroxyl group to prime the reverse transcription of the R2 RNA transcript. We have characterized the RNA requirement of this target DNA-primed reverse transcription reaction and found that the 250 nucleotides corresponding to the 3 untranslated region of the R2 transcript were necessary and sufficient for the reaction. To investigate the sequence requirements at the site of reverse transcription initiation, a series of RNA templates that contained substitutions and deletions at the extreme 3 end of the RNA were tested. The R2 templates used most efficiently had 3 ends which corresponded to the precise boundary of the R2 element with the 28S gene found in vivo. Transcripts containing short polyadenylated tails (8 nucleotides) were not utilized efficiently. R2 RNAs that were truncated at their 3 ends by 3 to 6 nucleotides were used less efficiently as templates and then only after the R2 reverse transcriptase had added extra, apparently nontemplated, nucleotides to the target DNA. The ability of the reverse transcriptase to add additional nucleotides to the target DNA before engaging the RNA template might be a mechanism for the generation of poly(A) or simple repeat sequences found at the 3 end of most non-long terminal repeatretrotransposable elements.Retrotransposable elements can be divided into two distinct groups on the basis of the presence or absence of terminal repeats and the nature of their encoded proteins (4,12,36,39,40). The first group to be identified consists of those elements that are flanked by long terminal repeats (LTR) and encode retrovirus-like gag and pol open reading frames. In agreement with their close structural similarity to retroviruses, all data on how this group of retrotransposable elements insert into eukaryotic genomes are consistent with a retrovirus-like mechanism (3, 30, 37). Indeed some LTR retrotransposable elements encode env-like genes and share certain transmission properties with retroviruses (23, 32).The second group of retrotransposable elements to be identified are not flanked by terminal repeats. A reverse transcriptase domain is the only homology that can be consistently identified for these elements (15). We will use the term non-LTR retrotransposable elements to identify this second group of elements (39). It has been demonstrated that these elements undergo retrotransposition by the removal of intron sequences during the generation of new copies (16,22,28). However, the absence both of LTRs and of an encoded integrase domain strongly suggests that these elements use a fundamentally different mechanism for their retrotransposition than that of the LTR-containing retrotransposable elements.Direct bioc...

L1 integration in a transgenic mouse model

“…L1 RT template jumping during a twin priming reaction likely explains the addition of 7 nt in another integrant (Supplemental Discussion 3). While to the best of our knowledge, RT template jumping during twin priming has not been reported previously, our results are consistent with the conceptually similar TPRT initiation, which occasionally creates 3Ј extra nucleotides due to the repeated attempts of RT to fully engage the RNA template (Luan and Eickbush 1995;Eickbush et al 2000;. The longer 47 extra-nucleotide addition was likely created by a series of three successive template jumps by L1 RT in the course of an otherwise unremarkable TPRT reaction (Fig.…”

“…While decapping of most cellular mRNAs requires prior shortening of the poly(A) tail (for review, see Coller and Parker 2004), we and others observed no difference between poly(A) lengths of 5Ј-truncated and FL integrants. This may imply that shortened L1 poly(A) tails are extended by the RT during TPRT initiation (Luan and Eickbush 1995;Eickbush et al 2000;Cost et al 2002) or are subject to additional polyadenylation after re-entry of L1 RNA into the nucleus.…”

HDV Family of Self-Cleaving Ribozymes

“…Target-primed reverse transcription and second-strand synthesis reconstruct the retrotransposon DNA for insertion at the new locus. 123,124 Until recently, the factor responsible for the cotranscriptional processing of the element was unknown, even though the processing was discovered over 25 years ago. 125 Recently, it has been confirmed that this event is due to the action of a self-cleaving ribozyme of an HDV-like motif (Fig.…”

Circular Noncoding Ribosomal Transcripts and Recombination of Retrotransposons: Two Charming Secrets Revealed by a Less-Explored Human Parasite

“…The latter would allow synthesis of both the complementary DNA and second strand DNA necessary to complete the element's integration starting from RNA. It is capable of adding nontemplate nucleotides (usually T residues), has high processivity (Luan and Eickbush, 1995), and low fidelity (Jamburuthugoda and Eickbush, 2011).Our preliminary work showed that the ORF2 protein encoded by EhLINE1 (recombinant fulllength ORF2p) has RT activity with various substrates, including poly (rA)-oligo(dT), and a 120 nt RNA template from the 3'-end of EhSINE1. EhLINE1-ORF2p seems to have considerably high processivity in vitro as full length cDNA was the major product with very few lower-sized products.…”

“…The latter would allow synthesis of both the complementary DNA and second strand DNA necessary to complete the element's integration starting from RNA. It is capable of adding nontemplate nucleotides (usually T residues), has high processivity (Luan and Eickbush, 1995), and low fidelity (Jamburuthugoda and Eickbush, 2011).…”